1. Field of the Invention
The present invention relates to astroviruses. In particular, the present invention relates to genomic and subgenomic nucleic acids of human astrovirus serotype 2.
2. Background Art
Astroviruses are 28-nm nonenveloped, viruses that were initially identified from the feces of infants with gastroenteritis by their distinctive ultrastructural features of characteristic five- or six-pointed surface stars (Appleton, J. et al., Lancet, 1: 1297 (1975); Madeley, C. R. et at., Lancet, 2: 451-452 (1975)). These nonenveloped agents were subsequently determined to be positive-strand RNA viruses (Herring, A. J. et al., J. Gen. Virol., 53: 47 (1981); Monroe, S. S. et at., J. Virol., 65: 641 (1991); Matsui, S. M. et al., J. Virol., 67: 1712 (1993)). Immune electron microscopy and immunofluorescence techniques have now identified five serotypes of human astroviruses, currently designated H-Ast1 to H-Ast5 (Kurtz, J. B. et al., Lancet, 2: 1405 (1984)).
Astroviruses cause acute gastroenteritis in children and adults worldwide (Cruz, J. R. et al., J. Clin. Microbiol., 30: 1140 (1992); Greenberg, H. B. et al., Infect. Agents Dis. 1: 71 (1992); Moe et al., J. Clin. Microbiol, 29: 2390 (1991)). However, the disease burden has been difficult to determine because of the lack of sensitive diagnostic assays. Recent studies have demonstrated that astroviruses were more frequently found in children with diarrhea than was previously thought (Herrmann et al., J. Infect. Dis. 161: 226 (1990); Herrmann et al., N. Engl. J. Med., 324: 1757 (1991); Lew, J. F. et al., J. Infect. Dis., 164: 673 (1991)). Outbreaks have been reported in kindergartens, (Konno, T. et al., J. Med. Virol., 9: 11-17 (1982)) pediatric wards (Kurtz, J. B. et al., J. Clin. Pathol., 30: 948-952 (1977)) and also in nursing homes (Gary, J. J. et al., J. Med. Virol., 23: 377-381 (1987); Oshiro, L. S. et al., J. Infect. Dis., 143: 791-795 (1981)).
Clinical signs associated with astrovirus infection include nausea, vomiting, non-bloody diarrhea, abdominal cramps, headaches, fever, chills and myalgia (LeBaron, C. W. et al., Morbidity and Mortality Weekly Report (Centers for Disease Control), Vol. 39 (Apr. 27, 1990)). Although most transmission is probably person-to-person among children, contaminated water and shellfish have also given rise to outbreaks in Britain (Kurtz, J. B. et at., (Ciba Foundation Symposium; 128), Chichester, UK: John Wiley & Sons Ltd., pp. 92-107 (1987)). Asymptomatic shedding of astrovirus has been documented (Ashley, C. R. et al., J. Clin. Pathol., 31: 939-943 (1978)) and infectivity can last as long as two days after clinical symptoms (White, K. E. et at., Am. J. Epiderniol., 124: 120-126 (1986)). Immuno-comprised individuals, e.g., AIDS patients, especially risk infection from astroviruses.
Previous studies of the biochemical properties of purified astrovirus particles have provided divergent results concerning the number and size of the proteins present in astroviruses; from two to as many as six polypeptides have been reported, ranging in size from 5.5 kDa to 42 kDa (Willcocks, M. M. et al., Rev. Med. Virol., 2: 97-106 (1992)). Likewise, there have been conflicting reports of the presence of subgenomic RNA present in astroviruses (Monroe, S. S. et al., J. of Virol., 65(2): 641-648 (1991); Willcocks et al., Arch. Virol., 124: 279-289 (1992). Moreover, characterization of the genome has been hindered because of the fastidious growth of astroviruses in vitro.
Investigators have reported partial sequence information from internal regions and at the 3' end of human astrovirus serotype 1 (H-Ast1) including: 1034 nucleotides from the 3' end of genomic RNA, (Willcocks, M. M. et al., Arch. Virol., 124: 279-289 (1992)) a 289 nucleotide immunoreactive epitope which overlaps the 3' end sequence (Matsui, S. M. et al., J. of Virol., 67: 1712-1715 (1993)), and two overlapping regions which hybridize only to genomic RNA (Matsui, S. M. et al. (1993)).
The fastidious nature of the virus coupled with extremely low levels of viral RNA generated by the organism during growth have made conventional sequencing approaches unpredictable and unreliable. Thus, despite a great need, the art has yet to provide sequences for human astrovirus type 2. The present invention satisfies this need by utilizing a unique combination of sequencing techniques to identify, diagnose, and treat astrovirus infection by providing nucleotide sequences for the complete genomic and subgenomic RNA of serotype 2 (H-Ast2) and analysis of the entire genomic RNA of H-Ast2. The present invention also provides the surprising discovery of a ribosomal frame shift occurring in one open reading frame which results in encoding of a fusional nonstructural polyprotein.